The role of Ca influx via the Na-Ca exchanger of the surface membrane (sarcolemma; SL) on activation of contraction is one of the most controversial issues in current studies on the cardiac excitation-contraction (E-C) coupling. Previous work has shown that the sarcoplasmic reticulum (SR), an intracellular Ca store and Ca transport system, is the major source of Ca which activates cardiac contraction, and plays a pivotal role in modulation of an impact of Ca influx via the SL Ca channels, (Ca current, Ica), ie. the prime trigger for SR Ca release. However, to what extent the SR buffers Ca influx via the Na-Ca exchange current (INaCa) and the impact of this on SR Ca release during a given beat is unknown. We recently developed an experimental protocol which enables simultaneous and quantitative assessment of Ca influx via INaCa, and of SR Ca loading and release, during a single cardiac beat. Intracellular Ca (Cai) transients (CAT) and membrane current are measured simultaneously in voltage-clamped, single ventricular myocytes dialyzed with the Ca indicator, indo-1. Ca influx selectively via INaCa is induced by voltage pulses from -40 to +90 mV, and graded by the pulse duration. Rapid exposures to caffeine (CAF), which open SR Ca release channels, are employed to deplete the SR of Ca prior to voltage-clamp stimulation (ST) and/or to abruptly release SR Ca accumulated during the same voltage pulse (ST+CAF) following SR Ca depletion. Under these conditions, configuration of CAT induced by ST+CAF reflects the total, net Ca influx via INaCa, while the CAT induced by ST, alone, reflects a portion of Ca influx which accesses the cytosol directly or via the SR. The difference in the CATs induced by ST+CAF and ST represents a portion of Ca influx via INaCa, which had been directly sequestered by the SR and not released to the cytosol during the same depolarization. We find that more than 80% of Ca influx via INaCa is directly buffered by the SR, and that the fractional release of accumulated Ca is greatly varied (from about 20 to 50%), by the resultant changes in SR Ca loading that occur during the same depolarization. Our results indicate that the SR plays a critical role in regulation of the effects of Ca influx via INaCa o cardiac E-C coupling.